The aim is to further define the molecular mechanisms of DNA replication initiation. Replication initiation is an essential event and is closely regulated in all biological systems. Replication fork formation and maintenance is critical to accurately duplicate genetic information for the next generation. In vivo structural analysis support a model in which the formation of a persistent RNA-DNA hybrid (R-loop) is an important intermediate in replication initiation. The pattern of initiation at T4 origins may serve as a basis for comparison of the structures and mechanisms of initiation at the origins of other prokaryotic and eukaryotic genomes. Origin plasmid topology will be used as a marker for RNA-DNA hybrid formation. Origin plasmid is extracted at early times after T4 phage infection and electrophoresed on two-dimensional chloroquine agarose gels with or without RNase H treatment. Any topological changes caused by the RNase H will be attributed to the removal of a persistent hybrid. Other related methods involve the use of topoisomerases to detect the RNA-DNA hybrid. The hybrid intermediate may serve to expose the nontemplate strand and the RNA itself as a primer.